1. The conserved aromatic palmsubdomain residues Tyr391 and Tyr619 interact with the last primertemplate basepair Tyr619 via a watermediated hydrogen bond with the phosphate of the terminal primer nucleotide and the mainchain amide of Tyr391 with the corresponding template nucleotide. A hydrogen bond has been postulated between Tyr391 and the hydroxyl group of Tyr567, a residue that plays a key role in base discrimination. This hydrogen bond may be crucial for forcing an infrequent Tyr567 rotamer conformation and, when the bond is removed, may influence fidelity. Tyr391 was replaced by Phe, Met and Ala, and Tyr619 by Phe. We used in vivo phage T4 rII reversion assays and an rI forward assay. The Y391A mutant, reported previously to decrease polymerase affinity for incoming nucleotides, was unable to support DNA replication in vivo, so for it we used an in vitro fidelity assay. Tyr391FM replacements affect fidelity only slightly, implying that the bond with Tyr567 is not essential for fidelity. The Y391A enzyme has no mutator phenotype in vitro. The Y619F mutant displays a complex profile of impacts on fidelity but has almost the same mutational spectrum as the parental enzyme. The Y619F mutant displays reduced DNA binding, processivity, and exonuclease activity on singlestranded and doublestranded DNA substrates. The Y619F substitution would disrupt the hydrogen bond network at the primer terminus and may affect the alignment of the 3 primer terminus at the polymerase active site, slowing chemistry and overall DNA synthesis.[unreadable] 2. The bindingpocket residue Tyr567 was shown previously to be a key determinant of fidelity, the replacement Y567A displaying a powerful mutator activity. Ser565 is a nearby component of the binding pocket. Unlike Y567A, the replacement S565G has only a small impact on fidelity. However, combining Y567A and S565G turns out to reverse most of the mutator activity of Y567A. This seems to be a unique and instructive observation in studies of polymerase fidelity, and we are midway through a detailed examination of the mutational propensities of these constructs both in vivo and in vitro. We are also pursuing structural insights into the S565Y567 interaction.